Project Summary Biomedical research and clinical diagnostics rely on the availability of high-quality diagnostic assays and sensors. The structure and functionality of biomolecular reagents used to construct these assays and sensors are highly dependent on storage conditions. However, due to the poor stability of these biomolecules (especially antibodies and enzymes), diagnostic assays and sensors must be maintained under refrigerated conditions to preserve their biofunctionality. Apart from causing financial and environmental burden, refrigeration or the ?cold chain? is simply not feasible in resource-limited settings such as rural clinics, battle fields, disaster-struck areas, low-income countries and regions with high burden of diseases and poor infrastructures. Therefore, to improve the accessibility of high-quality medical diagnostics in resource-limited settings, it is imperative to develop a refrigeration-free technology to provide stable and reliable bioassays and biosensors at the point-of-care. In this project, we will develop a novel technology that involves the use of metal-organic frameworks (MOFs) as encapsulants to preserve the functionality of biomolecular reagents in diagnostic assays and sensors under non- refrigerated conditions. The central hypothesis is that MOF encapsulated bioreagents can retain their biofunctionality under unregulated (fluctuating) temperature and humidity conditions, thus enabling the use of stable biosensors and bioassays in resource-limited settings. Our preliminary results have shown that MOF coatings can preserve surface-bound antibodies on plasmonic nanobiosensors against ambient and elevated temperatures. Two aims have been set to test the hypothesis. Aim 1 will develop and assess MOF coatings for preservation of surface-bound antibodies on plasmonic biosensors and enzyme-linked immunosorbent assays (ELISAs) under fluctuating temperature and humidity conditions. We will establish robust chemical procedures for MOF coatings on surface-bound antibodies on a model plasmonic nanobiosensor and ELISA, both will be used to detect neutrophil gelatinase-associated lipocalin (NGAL), a urinary biomarker for acute kidney injury. Aim 2 will develop and assess MOF/protein biocomposites for preservation of unbound (free) bioreagents including antibodies and enzymes used in ELISAs under fluctuating temperature and humidity conditions. We will focus on the preservation of two typical free biomolecular reagents used in sandwich ELISAs: the biotinylated detection antibody and streptavidin-horseradish peroxidase. Two different ELISAs for detection of urinary NGAL and serum prostate-specific antigen (PSA, a prostate cancer biomarker) will be selected as model systems. The successful completion of the proposed effort will lay the groundwork for a novel and generalizable MOF-based technology for preserving bioassays and biosensors, which not only eliminates refrigeration requirements, but also greatly advances the applications of various bioassay and biosensor platforms in point-of-care and resource-limited settings.